5-ht4 receptor agonist compounds for treatment of cognitive disorders

ABSTRACT

The invention provides methods for the treatment of cognitive disorders utilizing specific 5-HT 4  receptor agonist compounds, in particular, methods utilizing these compounds in combination with other agents, specifically acetylcholinesterase inhibitors, for the treatment of Alzheimer&#39;s disease and other cognitive disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos.61/168,741, filed on Apr. 13, 2009, and 61/292,559, filed on Jan. 6,2010, the disclosures of which are incorporated herein by reference intheir entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is directed to the use of specific 5-HT₄ receptor agonistcompounds for the treatment of cognitive disorders, in particular to theuse of these compounds in combination with other agents, specificallyacetylcholinesterase inhibitors for the treatment of Alzheimer's diseaseand other cognitive disorders.

2. State of the Art

The number of elderly people at risk of developing dementia is growingrapidly as life expectancy increases around the world. Alzheimer'sdisease is the most common cause of dementia in the elderly, accountingfor 50-60% of all cases, according to some experts. In 2008, anestimated 5.2 million people were living with Alzheimer's disease in theUnited States alone, accounting for 13% of the US population aged 65 andover.

Alzheimer's disease is defined as progressive cognitive decline andimpaired functional status inconsistent with normal aging. It isbelieved that deficits in the cholinergic system are a major contributorto the cognitive symptoms associated with Alzheimer's disease.Accordingly, the dominant pharmaceutical treatment for Alzheimer'sdisease provides modest symptomatic relief through the use ofacetylcholinesterase inhibitors. These agents are believed to act byreducing the rate of acetylcholine degradation thus leading to increasesin acetylcholine concentrations in the brain.

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter that iswidely distributed throughout the body, both in the central nervoussystem and in peripheral systems. At least seven subtypes of serotoninreceptors have been identified and the interaction of serotonin withthese different receptors is linked to a wide variety of physiologicalfunctions. The serotonergic system in the brain has been shown to beinvolved in cognitive processes. In particular, 5-HT₄ receptors havebeen demonstrated to play a role in the neuronal mechanism of memoryenhancement and cognitive processes in animal models. Activation of the5-HT₄ receptor enhances release of acetylcholine from cholinergicneurons, thus providing another potential approach to a pharmacologicalintervention that beneficially increases acetylcholine concentrations atsynapses within the brain (Maillet et al. (2004) Current AlzheimerResearch 1:79-85). Furthermore, it has been suggested that some 5-HT₄receptor agonist compounds may be used in the treatment of centralnervous system disorders including cognitive disorders, behavioraldisorders, mood disorders, such as depression and anxiety, and disordersof control of autonomic function.

Activation of the 5-HT₄ receptor also stimulates α-secretase activityresulting in increased levels of soluble amyloid precursor protein alpha(sAPPα) which has neurotrophic and neuroprotective properties, and isalso associated with cognitive enhancement preclinically. Beta amyloid(Aβ) is a peptide of 39-43 amino acids that appears to be the mainconstituent of amyloid plaques in the brains of Alzheimer's diseasepatients. Aβ is formed after cleavage of amyloid precursor protein by β-and γ-secretases. In preclinical studies, 5-HT₄ receptor agonist-inducedactivation of α-secretase, and generation of sAPPα, reduces the level ofAβ. Such a reduction in Aβ levels is expected to be beneficial.Therefore, 5-HT₄ receptor agonists offer the potential to provide bothsymptomatic and disease-modifying benefits (Lezoualc'h (2007)Experimental Neurology 205:325-329).

To date, no treatment that exploits the potential utility of the 5-HT₄mechanism for the treatment of cognitive disorders has been approved.Accordingly there remains a need for a treatment of memory dysfunctionin people suffering from Alzheimer's disease that takes advantage ofincreases in acetylcholine concentrations and other potential benefitsexpected from use of a 5-HT₄ receptor agonist agent.

SUMMARY OF THE INVENTION

The invention relates to the use of specific 5-HT₄ receptor agonistcompounds and to the use of specific 5-HT₄ receptor agonist compounds incombination with an acetylcholinesterase inhibitor for the treatment ofAlzheimer's disease or a cognitive disorder. In particular, theinvention relates to the use of the 5-HT₄ receptor agonist compounds andan acetylcholinesterase inhibitor, where each agent is used at aconcentration below the concentration at which a significant effect canbe observed when used alone.

The 5-HT₄ receptor agonist,1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide(1) and pharmaceutically-acceptable salts thereof

are disclosed in U.S. Pat. No. 7,375,114 B2. Compound 1 is alternativelydenoted as1,2-dihydro-N-[(3-endo)-8-[(2R)-2-hydroxy-3-[methyl(methylsulfonyl)amino]propyl]-8-azabicyclo[3.2.1]oct-3-yl]-1-(1-methylethyl)-2-oxo-3-quinolinecarboxamide.

The 5-HT₄ receptor agonist,4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester (2) and pharmaceutically-acceptable salts thereof

are disclosed in U.S. Pat. No. 7,256,294 B2 and US2006/0270652 A1.Compound 2 is alternatively denoted as4-[[4-[[[[2-(1-methylethyl)-1H-benzimidazol-7-yl]carbonyl]amino]methyl]-1-piperidinyl]methyl]-1-piperidinecarboxylicacid methyl ester.

Compounds 1 and 2 are potent and selective 5-HT₄ receptor agonists thatdemonstrate moderate to high intrinsic activity in in vitro assays.Compounds 1 and 2 have each been demonstrated to attenuate a muscarinicantagonist-induced memory deficit in the rat Morris water mazepreclinical cognition model. Results consistent with an additive orsynergistic effect were observed between compound 1 and theacetylcholinesterase inhibitor donepezil and between compound 2 anddonepezil at doses that on their own had little or no effect. Compounds1 and 2 have also been shown to evoke a concentration-dependent increasein sAPPu in in vitro assays. Compounds 1 and 2 are therefore expected tobe beneficial in the treatment of memory dysfunction.

In one aspect, the invention provides a method of treating Alzheimer'sdisease or a cognitive disorder in a patient, the method comprisingadministering to the patient a 5-HT₄ receptor agonist compound whereinthe compound is selected from1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amideand4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester, and pharmaceutically acceptable salts thereof.

The present 5-HT₄ agonist compounds are beneficially used in combinationwith an agent that acts to inhibit the action of acetylcholinesterase inthe body. Useful inhibitors include, but are not limited to, donepezilhydrochloride (available commercially as Aricept®), galantaminchydrobromide, alternatively written galanthamine hydrobromide(Razadyne®, Reminyl®), rivastigmine tartrate (Exelon®), and tacrinehydrochloride (Cognex®).

In another aspect, the invention provides a method of treatingAlzheimer's disease or a cognitive disorder in a patient, the methodcomprising administering to the patient a 5-HT₄ receptor agonistcompound wherein the compound is selected from1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amideand4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester, and pharmaceutically acceptable salts thereof and anacetylcholinesterase inhibitor.

In one aspect of the invention, in the above method, the 5-HT₄ agonistcompound and the acetylcholinesterase inhibitor are each administered ata dose that is subefficacious for treating Alzheimer's disease or acognitive disorder when administered alone.

In a specific aspect, the 5-HT₄ receptor agonist compound is1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amidehydrochloride and the acetylcholinesterase inhibitor is donepezil.

In another specific aspect, the 5-HT₄ receptor agonist compound is4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester and the acetylcholinesterase inhibitor is donepezil.

In yet another aspect, the invention provides a method of enhancingmemory in a patient experiencing a memory deficit, the method comprisingadministering to the patient a 5-HT₄ receptor agonist compound whereinthe compound is selected from1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amideand4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester, and pharmaceutically acceptable salts thereof and anacetylcholinesterase inhibitor.

In one aspect, the 5-HT₄ agonist compound and the acetylcholinesteraseinhibitor are each administered at a dose that is subefficacious forenhancing memory in a patient experiencing a memory deficit whenadministered alone.

As increase of sAPPα levels has been associated with cognitiveenhancement, in yet another aspect, the invention provides a method ofincreasing the levels of sAPPα in a patient, the method comprising (a)identifying a patient in need of increased production of sAPPα, i.e. apatient suffering from cognitive impairment, and (b) administering tothe patient a therapeutically-effective amount of a 5-HT₄ receptoragonist compound wherein the compound is selected from1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amideand4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester, and pharmaceutically acceptable salts thereof.

The invention further provides a pharmaceutical composition comprising apharmaceutically-acceptable carrier, a 5-HT₄ receptor agonist compound,wherein the compound is selected from1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amideand4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester, and pharmaceutically acceptable salts thereof, and anacetylcholinesterase inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the present invention are illustrated by reference tothe accompanying drawings.

FIGS. 1 through 6 display the mean escape latency in seconds on thethird test day of animals tested in the rat Morris water maze model ofcognitive function.

FIG. 1 shows results for administration of vehicle, scopolamine (Scop)(0.5 mg/kg), donepezil (0.3 mg/kg) plus scopolamine (0.5 mg/kg),donepezil (1 mg/kg) plus scopolamine (0.5 mg/kg), and donepezil (3mg/kg) plus scopolamine (0.5 mg/kg). * statistically significant withrespect to vehicle (student t-test with Bonferroni adjustment p<0.025) #statistically significant with respect to scopolamine (0.5 mg/kg)(one-way ANOVA for scopolamine (0.5 mg/kg) and all doses of donepezilplus scopolamine (0.5 mg/kg), post-hoc Dunnett's test p<0.05).

FIG. 2 shows results for administration of vehicle, scopolamine (Scop)(0.5 mg/kg), donepezil (3 mg/kg) plus scopolamine (0.5 mg/kg), compound1 (0.01 mg/kg) plus scopolamine (0.5 mg/kg), compound 1 (0.03 mg/kg)plus scopolamine (0.5 mg/kg), compound 1 (0.1 mg/kg) plus scopolamine(0.5 mg/kg) and compound 1 (0.1 mg/kg) plus scopolamine (0.5 mg/kg). ***statistically significant with respect to vehicle (student t-test withBonferroni adjustment p<0.0005) # statistically significant with respectto scopolamine (0.5 mg/kg) (one-way ANOVA for scopolamine (0.5 mg/kg)and all doses of compound 1 plus scopolamine (0.5 mg/kg), post-hocDunnett's test p<0.05).

FIG. 3 shows results for administration of vehicle, scopolamine (Scop)(0.5 mg/kg), GR125487 (1 mg/kg), compound 1 (0.1 mg/kg) plus GR125487 (1mg/kg) and scopolamine (0.5 mg/kg), and compound 1 (0.1 mg/kg) plusscopolamine (0.5 mg/kg). * statistically significant with respect tovehicle (student t-test with Bonferroni adjustment p<0.025) †\statistically significant with respect to compound 1 (0.1 mg/kg) plusGR125487 (1 mg/kg) and scopolamine (0.5 mg/kg) (student t-test withBonferroni adjustment p<0.005) # statistically significant with respectto scopolamine (0.5 mg/kg) (student t-test with Bonferroni adjustmentp<0.025).

FIG. 4 shows results for administration of vehicle, scopolamine (Scop)(0.5 mg/kg), donepezil (0.1 mg/kg) plus scopolamine (0.5 mg/kg),compound 1 (0.01 mg/kg) plus scopolamine (0.5 mg/kg), and compound 1(0.01 mg/kg) plus donepezil (0.1 mg/kg) and scopolamine (0.5 mg/kg) *statistically significant with respect to vehicle (student t-testp<0.05)

FIG. 5 shows results for administration of vehicle, scopolamine (Scop)(0.5 mg/kg), donepezil (3 mg/kg) plus scopolamine (0.5 mg/kg), compound2 (0.01 mg/kg) plus scopolamine (0.5 mg/kg), compound 2 (0.03 mg/kg)plus scopolamine (0.5 mg/kg), compound 2 (0.1 mg/kg) plus scopolamine(0.5 mg/kg) and compound 2 (1 mg/kg) plus scopolamine (0.5 mg/kg). ***statistically significant with respect to vehicle (student t-test withBonferroni adjustment p<0.0005) # statistically significant with respectto scopolamine (0.5 mg/kg) (one-way ANOVA for scopolamine (0.5 mg/kg)and all doses of compound 2 plus scopolamine (0.5 mg/kg), p=0.0096post-hoc Dunnett's test p<0.05).

FIG. 6 shows results for administration of vehicle, scopolamine (Scop)(0.5 mg/kg), donepezil (0.1 mg/kg) plus scopolamine (0.5 mg/kg),compound 2 (0.01 mg/kg) plus scopolamine (0.5 mg/kg), and compound 2(0.01 mg/kg) plus donepezil (0.1 mg/kg) and scopolamine (0.5 mg/kg) **statistically significant with respect to vehicle (student t-test withBonferroni adjustment p<0.005) # statistically significant with respectto scopolamine (0.5 mg/kg) (student t-test with Bonferroni adjustmentp<0.025).

DETAILED DESCRIPTION OF THE INVENTION Definitions

When describing the compositions and methods of the invention, thefollowing terms have the following meanings, unless otherwise indicated.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “subefficacious amount” or, equivalently, “subefficacious dose”means an amount or dose lower than a therapeutically effective amount ordose.

The term “treatment” as used herein means the treatment of a disease,disorder, or medical condition in a patient, such as a mammal(particularly a human) which includes, one or more of the following:

-   -   (a) preventing the disease, disorder, or medical condition from        occurring, i.e., prophylactic treatment of a patient;    -   (b) ameliorating the disease, disorder, or medical condition,        i.e., eliminating or causing regression of the disease,        disorder, or medical condition in a patient, including        counteracting the effects of other therapeutic agents;    -   (c) suppressing the disease, disorder, or medical condition,        i.e., slowing or arresting the development of the disease,        disorder, or medical condition in a patient; or    -   (d) alleviating the symptoms of the disease, disorder, or        medical condition in a patient.

The term “combination therapy” as used herein means the administrationof two or more therapeutic agents as part of a treatment protocolintended to provide beneficial effects from the combined action of thetherapeutic agents.

The term “acetylcholinesterase inhibitor” as used herein means any agentthat has the effect of inhibiting the action of acetylcholinesterase.The term includes agents termed cholinesterase inhibitors, which mayhave other activity in addition, for example as butyrylcholinesteraseinhibitors.

The term “donepezil” is used herein as an equivalent to donepezilhydrochloride.

Demonstration of Cognitive Enhancement In Vivo

As further described in the following examples, the efficacy of the5-HT₄ agonists, 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide(1) and4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester (2) in reversing muscarinic antagonist-induced memoryimpairment in rats was evaluated in the Morris water maze model.

Compound 1 at doses of 0.03, 0.1, and 1 mg/kg and compound 2 at doses of0.03 and 0.1 mg/kg attenuated the memory impairment induced in rats byinjection of scopolamine. For both compounds, a statisticallysignificant response was observed at 0.1 mg/kg. The potency and/orefficacy of both 5-HT₄ agonist compounds appeared to be similar to thatof donepezil, an acetylcholinesterase inhibitor marketed for thesymptomatic treatment of Alzheimer's disease. The cognitive enhancingeffects of compound 1 and of compound 2 were prevented bycoadministration of each compound with a selective 5-HT₄ antagonist,confirming involvement of 5-HT₄ receptors in the observed response.

Significantly, an additive or synergistic effect was noted betweencompound 1 and donepezil at doses of 0.01 mg/kg and 0.1 mg/kg,respectively, and between compound 2 and donepezil at doses of 0.01mg/kg and 0.1 mg/kg, respectively, doses that were not effective atreversing scopolamine-induced cognitive decline when administered alone.

Demonstration of sAPPα Release In Vitro

The effects of compounds 1 and 2 on extracellular release of sAPPα wasstudied in HEK293 cells stably-transfected with human 5-HT_(4(d))receptors and human APP₆₉₅ (amyloid precursor protein). Both compoundsevoked a concentration-dependent increase in sAPPα release. The releasewas blocked by the 5-HT₄ receptor-selective antagonist GR113808,indicating that the observed effect is related to agonism of the 5-HT₄receptor.

Methods of Treatment

The present 5-HT₄ agonists, compounds 1 and 2, are expected to be usefulfor the treatment of Alzheimer's disease or a cognitive disorder,including treatment of mild cognitive impairment, and treatment ofmemory dysfunction associated with Alzheimer's disease and of dementiaof the Alzheimer's type. The compounds may further find utility in thetreatment of additional central nervous system disorders includingbehavioral disorders, mood disorders, such as depression and anxiety,and disorders of control of autonomic function. In addition, it has beensuggested that compounds that enhance acetylcholine concentrations mayalso be useful for the treatment of other forms of dementia, such asdementia associated with Parkinson's disease, dementia due to vascularmechanisms, and Lewy body dementia.

An additive or synergistic effect was noted in the rat water mazeexperiments when the present 5-HT₄ compounds were coadministered with anacetylcholinesterase inhibitor at doses where each compound wasineffective when administered alone. Accordingly, the present compoundsare expected to be useful for the treatment of Alzheimer's disease or acognitive disorder, when administered in combination with anacetylcholinesterase inhibitor such as donepezil hydrochloride(Aricept®), galantamine hydrobromide, (Razadyne®, Reminyl®),rivastigmine tartrate (Exelon®), or tacrine hydrochloride (Cognex®). Inaddition to providing the benefit of agents acting by different,complementary mechanisms of action, combination therapy offers theadditional potential benefit of allowing lower dosages of each agent tobe used, thus limiting exposure to any adverse side effects.

Further, compounds 1 and 2 may be useful when coadministered with agentsdesigned to provide symptomatic therapy to Alzheimer's patients by yetother mechanisms of action. For example the present compounds may beuseful in combination with memantine (Namenda®), an NMDA receptorantagonist. Additional agents that may be used in combination for thetreatment of Alzheimer's disease include 5-HT₆ antagonists such asDMXB-anabaseine, GSK-742457, SUVN-502, PRX-07034, and SAM-531(WAY-262531); nicotinic receptor agonists, such as ABT-089, SSR-180711,AZD-0328, and EVP-6124; muscarinic M₁ agonists, such as NGX-267, AF-102B(Cevimeline), and WAL 2014 FU (talsaclidine); histamine H₃ antagonistssuch as GSK-189254 and PF-365474; and dimebon. Also, 5-HT₄ agonists maybe beneficial in combination with other proposed disease modifyingtherapies such as amyloid-beta and tau aggregation inhibitors, and betasecretase inhibitors or gamma secretase inhibitors, such as BMS-708163.

When used in combination therapy, the present 5-HT₄ compounds are eitherphysically mixed with the other therapeutic agent to form a compositioncontaining both agents; or each agent is present in separate anddistinct compositions which are administered to the patientsimultaneously or sequentially in any order. Combination therapyincludes administration of the two agents, when formulated separately,substantially at the same time, as well as administration of each agentat a different time.

For example, the present 5-HT₄ compounds can be combined with a secondtherapeutic agent using conventional procedures and equipment to form acomposition comprising compound 1 or compound 2 and a second therapeuticagent. Additionally, the therapeutic agents may be combined with apharmaceutically acceptable carrier to form a pharmaceutical compositioncomprising compound 1 or compound 2, a second therapeutic agent, and apharmaceutically acceptable carrier. In this embodiment, the componentsof the composition are typically mixed or blended to create a physicalmixture. The physical mixture is then administered in a therapeuticallyeffective amount using any of the routes described below.

Alternatively, the therapeutic agents may remain separate and distinctbefore administration to the patient. In this embodiment, the agents arenot physically mixed together before administration but are administeredsimultaneously or at separate times as separate compositions. Suchcompositions can be packaged separately or may be packaged together as akit. The two therapeutic agents in the kit may be administered by thesame route of administration or by different routes of administration.

When used to treat Alzheimer's disease or cognitive disorders, compound1 or compound 2 will typically be administered orally in a single dailydose or in multiple doses per day, although other forms ofadministration may be used. In certain circumstances, it may bebeneficial to administer the present 5-HT₄ compounds transdermally orparenterally. The amount of active agent administered per dose or thetotal amount administered per day will typically be determined by aphysician, in the light of the relevant circumstances, including theseverity of the condition to be treated, the chosen route ofadministration, the specific compound administered and its relativeactivity, the age, weight, and response of the individual patient, andthe like.

Suitable doses for treating Alzheimer's disease or cognitive disorderswill range from about 0.1 to about 90 mg per day of 5-HT₄ agonist agentfor an average 70 kg human, including, for example, from about 1 toabout 50 mg per day of compound 1, and from about 0.5 to about 20 mg perday of compound 2.

When the present 5-HT₄ compounds are used in combination therapy with anacetylcholinesterase inhibitor, the acetylcholinesterase inhibitor isadministered in a therapeutically effective amount, i.e. in any amountthat produces a therapeutically beneficial effect when co-administeredwith compound 1 or compound 2. Suitable doses for theacetycholinesterase inhibitor administered in combination with thepresent compounds are typically in the range of about 1 mg/day to about30 mg/day. As described previously, when used as part of combinationtherapy, an effective dose of each individual agent may be lower thanthe effective dose when the agents are used independently.

Pharmaceutical Compositions

The present 5-HT₄ agonists and the other therapeutic agents, such asacetylcholinesterase inhibitors, are typically administered to a patientin the form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to, oral,rectal, vaginal, nasal, inhaled, topical (including transdermal) andparenteral modes of administration.

The pharmaceutical compositions typically contain a therapeuticallyeffective amount of the active agents. Those skilled in the art willrecognize, however, that a pharmaceutical composition may contain morethan a therapeutically effective amount, i.e., bulk compositions, orless than a therapeutically effective amount, i.e., individual unitdoses designed for multiple administration to achieve a therapeuticallyeffective amount.

Typically, such pharmaceutical compositions will contain from about 0.1to about 95% by weight of the active agent; preferably, from about 5 toabout 70% by weight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the carriers or excipients used inthe pharmaceutical compositions of this invention arecommercially-available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with apharmaceutically-acceptable carrier and one or more optionalingredients. The resulting uniformly blended mixture can then be shapedor loaded into tablets, capsules, pills and the like using conventionalprocedures and equipment.

The pharmaceutical compositions are preferably packaged in a unit dosageform. The term “unit dosage form” refers to a physically discrete unitsuitable for dosing a patient, i.e., each unit containing apredetermined quantity of active agent calculated to produce the desiredtherapeutic effect either alone or in combination with one or moreadditional units. For example, such unit dosage forms may be capsules,tablets, pills, and the like, or unit packages suitable for parenteraladministration.

In one embodiment, the pharmaceutical compositions of the invention aresuitable for oral administration. Suitable pharmaceutical compositionsfor oral administration may be in the form of capsules, tablets, pills,lozenges, cachets, dragees, powders, granules; or as a solution or asuspension in an aqueous or non-aqueous liquid; or as an oil-in-water orwater-in-oil liquid emulsion; or as an elixir or syrup; and the like;each containing a predetermined amount of a therapeutic compound as anactive ingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionswill typically comprise the active agent and one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate. Optionally or alternatively, such solid dosageforms may also comprise: fillers or extenders, such as starches,microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/orsilicic acid; binders, such as carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, suchas glycerol; disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and/or sodium carbonate; solution retarding agents, such as paraffin;absorption accelerators, such as quaternary ammonium compounds; wettingagents, such as cetyl alcohol and/or glycerol monostearate; absorbents,such as kaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of the invention. Examples ofpharmaceutically-acceptable antioxidants include: water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate,alpha-tocophcrol, and the like; and metal-chelating agents, such ascitric acid, ethylenediamine tetraacetic acid, sorbitol, tartaric acid,phosphoric acid, and the like. Coating agents for tablets, capsules,pills and like, include those used for enteric coatings, such ascellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, methacrylic acid-methacrylic acid estercopolymers, cellulose acetate trimellitate, carboxymethyl ethylcellulose, hydroxypropyl methyl cellulose acetate succinate, and thelike. Coating agents also include talc, polyethylene glycol, hypomelloseand titanium dioxide.

Pharmaceutical compositions may also be formulated to provide slow orcontrolled release of the active agent using, by way of example,hydroxypropyl methyl cellulose in varying proportions; or other polymermatrices, liposomes and/or microspheres. In addition, pharmaceuticalcompositions may optionally contain opacifying agents and may beformulated so that they release the active ingredient only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes. The activeagent can also be in micro-encapsulated form, if appropriate, with oneor more of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (esp., cottonseed, groundnut, corn, germ, olive, castor andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Suspensions, inaddition to the active ingredient, may contain suspending agents suchas, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The present active agents can also be administered parenterally (e.g. byintravenous, subcutaneous, intramuscular or intraperitoneal injection).For parenteral administration, the active agent is typically admixedwith a suitable vehicle for parenteral administration including, by wayof example, sterile aqueous solutions, saline, low molecular weightalcohols such as propylene glycol, polyethylene glycol, vegetable oils,gelatin, fatty acid esters such as ethyl oleate, and the like.Parenteral formulations may also contain one or more anti-oxidants,solubilizers, stabilizers, preservatives, wetting agents, emulsifiers,buffering agents, or dispersing agents. These formulations may berendered sterile by use of a sterile injectable medium, a sterilizingagent, filtration, irradiation, or heat.

Alternatively, the agents are formulated for administration byinhalation. Suitable pharmaceutical compositions for administration byinhalation will typically be in the form of an aerosol or a powder. Suchcompositions are generally administered using well-known deliverydevices, such as a metered-dose inhaler, a dry powder inhaler, anebulizer or a similar delivery device.

When administered by inhalation using a pressurized container, thepharmaceutical compositions of the invention will typically comprise theactive ingredient and a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas.Additionally, the pharmaceutical composition may be in the form of acapsule or cartridge (made, for example, from gelatin) comprising acompound of the invention and a powder suitable for use in a powderinhaler. Suitable powder bases include, by way of example, lactose orstarch.

Finally, active agents can also be administered transdermally usingknown transdermal delivery systems and excipients. For example, theactive agent can be admixed with permeation enhancers, such as propyleneglycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and thelike, and incorporated into a patch or similar delivery system.Additional excipients including gelling agents, emulsifiers and buffers,may be used in such transdermal compositions if desired.

Representative pharmaceutical compositions useful for the treatment ofAlzheimer's disease or a cognitive disorder, include, but are notlimited to, the following examples where ‘compound of the invention’represents compound 1 or compound 2. Compound 1 is typically supplied asa hydrochloride salt and compound 2 is typically supplied as a freebase, but it will be understood that any form of the compounds (i.e.free base or pharmaceutical salt) that is suitable for the particularmode of administration, can be used in the following pharmaceuticalcompositions.

Formulation Example A Hard Gelatin Capsules for Oral Administration

A compound of the invention (20 mg), starch (89 mg), microcrystallinecellulose (89 mg), and magnesium stearate (2 mg) are thoroughly blendedand then passed through a No. 45 mesh U.S. sieve. The resultingcomposition is loaded into a hard gelatin capsule (200 mg of compositionper capsule).

Formulation Example B Gelatin Capsules for Oral Administration

A compound of the invention (10 mg), polyoxyethylene sorbitan monooleate(50 mg), and starch powder (250 mg) are thoroughly blended and thenloaded into a gelatin capsule (310 mg of composition per capsule).

Formulation Example C Tablets for Oral Administration

A compound of the invention (5 mg), microcrystalline cellulose (400 mg),fumed silicon dioxide (10 mg), and stearic acid (5 mg) are thoroughlyblended and then compressed to form tablets (420 mg of composition pertablet).

Formulation Example D Tablets for Oral Administration

A compound of the invention (2 mg), microcrystalline cellulose (400 mg),fumed silicon dioxide (10 mg), and stearic acid (5 mg) are thoroughlyblended and then compressed to form tablets (417 mg of composition pertablet).

Formulation Example E Tablets for Oral Administration

A compound of the invention (20 mg), microcrystalline cellulose (400mg), fumed silicon dioxide (10 mg), and stearic acid (5 mg) arethoroughly blended and then compressed to form tablets (435 mg ofcomposition per tablet).

Formulation Example F Single-Scored Tablets for Oral Administration

A compound of the invention (15 mg), cornstarch (50 mg), croscarmellosesodium (25 mg), lactose (120 mg), and magnesium stearate (5 mg) arethoroughly blended and then compressed to form single-scored tablet (215mg of compositions per tablet).

Formulation Example G Suspension for Oral Administration

The following ingredients are thoroughly mixed to form a suspension fororal administration containing 20 mg of active ingredient per 10 mL ofsuspension: compound of the invention (200 mg), sodium benzoate, sodiumcitrate, purified water (q.s. to 100 mL).

Formulation Example H Injectable Formulation

A compound of the invention (20 mg) is blended with 0.1 M sodium citratebuffer solution (15 mL). The pH of the resulting solution is adjusted topH 6 using 1 N aqueous hydrochloric acid or 1 N aqueous sodiumhydroxide. Sterile normal saline in citrate buffer is then added toprovide a total volume of 20 mL.

Formulation Example I Single-Scored Tablets for Oral Administration

A compound of the invention (5 mg), donepezil hydrochloride (5 mg),cornstarch (50 mg), microcrystalline cellulose (15 mg), hydroxypropylcellulose (10 mg) lactose (120 mg), and magnesium stearate (5 mg) arethoroughly blended and then compressed to form single-scored tablet (210mg of compositions per tablet).

Formulation Example J Suspension for Oral Administration

The following ingredients are thoroughly mixed to form a suspension fororal administration containing 5 mg of each agent per 10 mL ofsuspension: compound of the invention (50 mg), rivastigminc tartrate (50mg) sodium benzoate, sodium citrate, purified water (q.s. to 100 mL).

EXAMPLES

The ability of the 5-HT₄ agonists,1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide(1) and4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester and (2), used alone or in combination with theacetylcholinesterase inhibitor, donepezil, to prevent spatial memoryimpairment induced by the muscarinic receptor antagonist, scopolamine,was evaluated in the rat Morris water maze animal model (Morris (1984)Journal of Neuroscience Methods 11:47-60.). The hydrochloride salt ofcompound 1 was used in all experiments.

Morris Water Maze Model Methodology

Adult, male Sprague-Dawley rats (body weight range 275-400 g) werehandled by the investigator for approximately 5 minutes one day prior toeach study. On the first study day, each rat was dosed intraperitoneally(i.p.), first with test compound or vehicle, followed immediatelythereafter with the muscarinic receptor antagonist, scopolamine (0.5mg/kg), a dose previously identified to provide a near-maximal cognitivedecline in this model) or vehicle. Thirty minutes after dosing, ratswere placed individually into a circular, dark blue polyethylene tank (6feet in diameter) filled with water (maintained at 23° C.±1° C.). Aclear Perspex® platform was located in a fixed position 1 cm below thesurface of the water, and 34 cm from the wall of the tank. Four visualcues (A4-sized pictures of different black and white symbols) wereequally spaced on the wall of the tank, just above the water surface.Black curtains were placed around the tank and white noise was generatedthroughout the test. Each rat was released into the water, facing thetank wall, at a designated starting point (i.e., at the north side ofthe tank), and allowed 60 seconds to locate the submerged platform aftereach placement. The escape latency (in seconds) between release of therat into the water and its locating the platform was recordedautomatically by means of a video camera (San Diego Instrument, CA) andtracking software (SMART software, San Diego Instrument, CA). When a ratfailed to find the platform within 60 seconds, it was guided towards theplatform and then placed on it (facing a specific visual cue). The ratswere allowed to remain on the platform for 30 seconds to observe theexternal visual cues and pair them with the relative position of theplatform. The rats were then taken off the platform and dried gentlywith a paper towel before repeating the test three more times (fromsouth, then east and finally west starting positions). After the fourthtest, each rat was placed under a heating lamp for 5 minutes beforebeing returned to its home cage. The entire procedure was repeated ondays 2 and 3. Over the three days of testing, rats learned to associatethe location of the submerged platform (and their only means to escapethe water) with the visual cues.

The ability of test agents to inhibit scopolamine-induced cognitiveimpairment was evaluated. On day 4, each rat was dosed as before andsubjected to a 2-minute “probe trial”, for which the platform had beenremoved from the tank. The number of crossings of the previous platformlocation and the time spent by the rat in each quadrant were recorded.Animals that had learned the location of the platform spent more time inthe appropriate quadrant and repeatedly crossed that quadrant. Byperforming the probe test, false positives could be excluded from theanalysis (i.e., rats that had previously located the platform via astrategy independent of the visual cues, such as swimming randomly untila leg hit the platform). No animals were excluded based on conflictingdata between the three days of testing and the probe test.

Data Analysis

The average escape latency from each of the four trials on the same testday was calculated for individual rats, and then these data werecombined to determine the mean escape latency for each treatment groupon days 1, 2 and 3. The following statistical tests were performed onthe data:

A student's t-test compared the escape latencies of the vehicle/vehicleand vehicle/scopolamine groups. The cc value was Bonferroni adjusted to0.025 as the vehicle/scopolamine group was subjected to both a Student'st-test and a one-way ANOVA test.

A one-way ANOVA, followed by a Dunnett's post-hoc test, was used tocompare the escape latencies of the vehicle/scopolamine and testcompound/scopolamine groups, with p<0.05 indicating a statisticallysignificant difference.

A two-way repeated-measures ANOVA, followed by a Bonferroni post-hoctest, was used to compare the mean escape latency for each of the threesuccessive test days, with p<0.05 indicating a statistically significantdifference.

Materials

The 5-HT₄ agonist compounds 1 and 2 were prepared according to theprocedures described in U.S. Pat. No. 7,375,114 B2 and U.S. Pat. No.7,256,294 B2, respectively, the disclosures of which are incorporatedherein by reference. Donepezil hydrochloride was purchased fromChangzhou Dahua Imp. and Exp. Corp. Ltd. (Changzhou, Jiangsu, China)while scopolamine hydrochloride was purchased from Sigma Aldrich (St.Louis, Mo.) or Spectrum Chemical Mfg. Corp. (Gardena, Calif.). GR125487sulfamate was purchased from Tocris (Ellisville, Mo.). Compounds 1 and 2and donepezil were formulated in 5% dimethyl sulfoxide and 95% sterilesaline, while scopolamine and GR125487 were prepared in 100% sterilesaline. Doses were expressed with respect to the free base weights ofeach compound.

Model Validation

In each study, dosing (i.p.) of rats with vehicle 30 min prior to thetest resulted in a progressive reduction in escape latency. On day 1,vehicle-treated rats typically located the hidden platform with a meanlatency from the four trials in the range of 40-50 seconds, while on day3, this had shortened to 10-25 seconds. Scopolamine (0.5 mg/kg i.p.)produced a statistically significant attenuation of learning (p<0.025,Student's t-test with Bonferroni's adjustment) as compared with vehicletreated animals. The acetylcholinesterase inhibitor donepezil (3 mg/kgi.p.), and the 5-HT₄ receptor agonists, compound 1 (1 mg/kg i.p.) andcompound 2 (1 mg/kg i.p.), had no effect, either positive or negative,on the ability of rats to learn the location of the submerged platformwhen administered alone, i.e. when administered to animals that had notbeen exposed to scopolamine. Donepezil (0.3-3 mg/kg i.p.), reversed thescopolamine (0.5 mg/kg i.p.)-induced cognitive deficit in adose-dependent manner as shown in FIG. 1 where the mean escape latencyon the third test day is displayed. The donepezil dose at 3 mg/kgproduced a statistically significant reversal.

Example 1 The Effect of Compound 1 on Scopolamine-Induced CognitiveImpairment

Compound 1 was tested at doses of 0.01, 0.03, 0.1, and 1 mg/kg in therat Morris Water Maze model. As shown in FIG. 2, where the mean escapelatency on the third test day is displayed, compound 1 was associatedwith reversal of the memory impairment produced by 0.5 mg/kgscopolamine. The effect of compound 1, at a dose of 0.1 mg/kg, achievedstatistical significance with respect to scopolamine (0.5 mg/kg). A“U-shaped” dose-response curve was evident; the highest dose of compound1, 1 mg/kg, had no effect on the scopolamine-induced response unlike the0.1 mg/kg dose. Compound 1 (0.1 mg/kg i.p.) produced a similar reversalto that of donepezil (3 mg/kg i.p.) on day 3 (i.e., in the range of50-60%, see FIG. 1)

Example 2 The Effect of Compound 1 Together with a 5-HT₄ ReceptorAntagonist on Scopolamine-Induced Cognitive Impairment

In order to probe whether the observed effects of compound 1 can beattributed to agonism at the 5-HT₄ receptor, compound 1 was testedtogether with GR125487, a selective 5-HT₄ receptor antagonist compound.As shown in FIG. 3, GR125487 (1 mg/kg i.p.) had no effect whenadministered alone. However, coadministration of the antagonist GR125487(1 mg/kg i.p.) abolished the ability of compound 1 (0.1 mg/kg i.p.) toreverse scopolamine-induced cognitive impairment. Therefore, it isreasonable to conclude the observed effects of compound 1 are due to5-HT₄ receptor agonism.

Example 3 The Effect of Co-Administration of Compound 1 Together with athe Acetylcholinesterase Inhibitor Donepezil on Scopolamine-InducedCognitive Impairment

The effect of co-administration of compound 1 and donepezil isillustrated in FIG. 4. Neither compound 1 at a dose of 0.01 mg/kg nordonepezil at a dose of 0.1 mg/kg were found to have a significant effectwhen administered alone. However, the same doses of compound 1 anddonepezil, when formulated together in 5% DMSO and 95% sterile saline,resulted in a noticeable attenuation of the scopolamine-inducedcognitive decline.

Example 4 The Effect of Compound 2 on Scopolamine-Induced CognitiveImpairment

Compound 2 was tested as described in Example 1. As illustrated in FIG.5, the effect of compound 2 at a dose of 0.1 mg/kg, achieved statisticalsignificance with respect to scopolamine. A “U-shaped” dose-responsecurve was evident; the highest dose of compound 2, 1 mg/kg, had noeffect on the scopolamine-induced response unlike the 0.03 and 0.1 mg/kgdoses. Compound 2 (0.1 mg/kg i.p.) produced a similar reversal to thatof donepezil (3 mg/kg i.p.) on day 3 (i.e., in the range of 50-60%, seeFIG. 1).

Example 5 The Effect of Compound 2 Together with a 5-HT₄ ReceptorAntagonist on Scopolamine-Induced Cognitive Impairment

The effect of co-administration of compound 2 with the 5-HT₄ receptorantagonist GR125487 was probed as described in Example 2. AddingGR125487 (1 mg/kg i.p.) abolished the ability of compound 2 (0.1 mg/kgi.p.) to reverse scopolamine-induced cognitive impairment, whichindicates the observed effects of compound 2 are due to 5-HT₄ receptoragonism.

Example 6 The Effect of Co-Administration of Compound 2 Together with athe Acetylcholinesterase Inhibitor Donepezil on Scopolamine-InducedCognitive Impairment

The effect of co-administration of compound 2 and donepezil isillustrated in FIG. 6. Neither compound 2 at a dose of 0.01 mg/kg nordonepezil at a dose of 0.1 mg/kg were found to have a significant effectwhen administered alone. However, the same doses of compound 2 anddonepezil, when formulated together in 5% DMSO and 95% sterile saline,resulted in a statistically significant reversal (student t-test withBonferroni adjustment p<0.025) of the scopolamine-induced cognitivedecline.

Example 7 In Vitro Study of Effects of Compound 1 and Compound 2 onExtracellular Release of sAPPα in HEK293-5-HT_(4(d))-APP₆₉₅ Cells

Cell Culture

HEK-293 (human embryonic kidney) cells stably-transfected with human5-HT_(4(d)) receptor cDNA and human APP₆₉₅ cDNA(HEK293-5-HT_(4(d))-APP₆₉₅) were grown in Dulbecco's Modified EaglesMedium (DMEM) containing D-glucose supplemented with 10% fetal bovineserum, 2 mM GlutaMax-1, and 100 units penicillin (100 μg), and 100 μg/mLstreptomycin in a 5% CO₂, humidified incubator at 37° C. Cells weregrown under continuous selection pressure by the addition of G418 (500ug/mL) antibiotic.

sAPPα Release

HEK293-5-HT_(4(d))-APP_(o95) cells (3×10⁵ cells/well) were serum-starvedfor 4 hr prior to incubation with agonists for 30 min (unless otherwisestated) at 37° C. The culture medium was aspirated, centrifuged toremove cellular debris and the level of sAPPα determined by Westernblot. sAPPα was detected using antibody 6E10 (Signet/Covance) and a goatanti-mouse HRP-conjugated 2° antibody. Immunoreactive bandscorresponding to sAPPα were visualized and quantified with ECL substrate(Pierce) and a Fluor Chem HD2 image system (Alpha Innotech). Westernblot analysis of samples was conducted in duplicate.

Results

Potency data are reported as pEC₅₀ values, the negative decadiclogarithm of the EC₅₀ value, where EC₅₀ is the effective concentrationfor a 50% maximal response. Test compounds exhibiting a higher pEC₅₀value in this assay have a higher potency for stimulating sAPPα release.To determine EC₅₀ values, data from independent experiments were fitsimultaneously to a sigmoidal concentration response curve using GraphPad Prism software (slope constrained to unity). Potency data forcompound 1 and compound 2, together with the percentage responserelative to the effect of the endogenous ligand 5-HT at a concentrationof 1 μM are given below:

E_(max) pEC₅₀ (% response relative to 1 μM 5-HT) Compound 1 8.2 91Compound 2 9.0 103

The effect of the 5-HT₄ receptor-selective antagonist GR113808 wasprobed by incubating the cells for 10 min with 1 μM GR113808 beforeincubating with compound 1 or compound 2 at a concentration of 100 nM, aconcentration at which the compounds evoked a near-maximal or maximalresponse. The antagonist completely blocked the effect of compound 1 andof compound 2, indicating that the observed release of sAPPα is relatedto agonism of the 5-HT₄ receptor.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. Additionally, all publications, patents, andpatent documents cited hereinabove are incorporated by reference hereinin full, as though individually incorporated by reference.

1-13. (canceled)
 14. A pharmaceutical composition comprising anacetylcholinesterase inhibitor, a 5-HT₄ receptor agonist compoundwherein the compound is selected from1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amideand4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester, and pharmaceutically acceptable salts thereof, and apharmaceutically-acceptable carrier.
 15. The pharmaceutical compositionof claim 14 wherein the acetylcholinesterase inhibitor is donepezilhydrochloride.
 16. The pharmaceutical composition of claim 14 whereinthe 5-HT₄ receptor agonist compound is1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide,or a pharmaceutically-acceptable salt thereof.
 17. The pharmaceuticalcomposition of claim 14 wherein the 5-HT₄ receptor agonist compound is1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amidehydrochloride.
 18. The pharmaceutical composition of claim 14 whereinthe 5-HT₄ receptor agonist compound is4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester. 19-20. (canceled)